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Figure 3. myo1d Is Required for GRP Morphogenesis and Leftward Flow
(A–E) GRP ciliation. Dorsal explants were prepared and analyzed for the presence and polarization of cilia by immunofluorescence using an antibody against acetylated alpha-tubulin. Counterstaining of actin using Phalloidin highlighted cell boundaries.
(A) Wild-type (blow-up shown in B).
(C) myo1d morphant.
(D and E) Blowups of severe phenotype shown in (D) and of moderate phenotype shown in (E).
(F–J) Quantification of cilia lengths (F), ciliation rate (G), cilia polarization (H), flow velocity (I), and flow directionality (J).
(K and L) Wild-type expression of nodal1 in control (K) and myo1d morphant (L) stage 19 embryo.
(M–O) Asymmetrical dand5 expression in lateral GRP cells of wild-type control embryo (M) was lost in myo1d morphant specimen (N).
(O) Quantification of dand5 expression patterns.
(K)–(N) are shown at the same magnification.
Numbers represent analyzed specimens, which were derived from 3 (A–H), 2 (I and J), and 5 (K–O) independent experiments. For the assessment of cilia polarization, 15 cilia were analyzed per explant, for cilia lengths 30 cilia per GRP, and the ciliation rate was determined upon evaluating the entire GRP. |
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Figure S1. Myo1d is required for LR axis formation, Related to Figure 1.
(A-H) nodal1 (A-D) and pitx2 (E-H) expression in wildtype (A, E) and myo1d morphant (B-D, F-H) embryos, as
determined by WM-ISH with antisense probes for nodal1 (A-D) and pitx2 (E-H).
(I, J) Downregulation of Myo1d protein in myo1d morphants. (I) Tadpole lysates were probed with Mab4E12 before
(left) and after (right) affinity purification (ap). (J) Embryos were injected at the 2-4 cell stage with 1 ng of AUG-MO or
an antisense MO containing 5 mismatches (MM-MO). Affinity purified Mab4E12 was used to probe western blots
containing lysates from stage 28 embryos.
(K, L) Lefty1 mRNA expression in wildtype (K) and myo1d morphant (L) specimen, as shown by transversal
histological sections of WM-ISH stained embryos at stage 24. fp, floor plate; hc, hypochord; MM-MO, mismatch MO. |
